Project Summary/Abstract Melanoma is a highly fatal malignancy for which immunotherapy approaches have been shown to be effective. While immune checkpoint blockade (ICB) has transformed the treatment of advanced melanoma, the majority of patients are resistant to therapy initially or respond but then relapse. Thus, efforts to identify mechanisms of resistance to checkpoint inhibitors and approaches to overcome primary and acquired resistance are warranted. Surprisingly, type II interferon (IFN?) signaling pathways and expression of IFN-stimulated genes (ISGs) in melanoma patients have been shown to correlate with either response or resistance to ICB treatment, in a context-dependent manner. While IFN?-inducible genes are required for effector function of tumor-reactive T cells and response to ICB in melanoma, IFN? is also known to induce expression of immunosuppressive genes and chronic IFN? signaling promotes acquired resistance to ICB. This dual role of IFN? signaling supports the need to identify means to selectively regulate its effects in order to promote cytotoxic T lymphocyte (CTL) activity without promoting immunosuppression. We have recently discovered that ULK1 (Unc-51-like kinase 1) controls activation of unique IFN? signaling events and transcription of specific IFN?-induced genes involved in the control of immune responses. Importantly, our preliminary studies show that genetic or pharmacologic targeting of ULK1 in melanoma cells represses IFN?-induced expression of immunosuppressive genes without affecting expression of immunostimulatory ones, and high levels of ULK1 expression correlate with poor survival in melanoma patients. The goal of this project is to determine if targeting ULK1 blocks IFN?-dependent immunosuppressive effects and related biological responses, while promoting IFN?-mediated CTL activity against melanoma. Aim 1 will define the specific role of ULK1 on IFN?-induced expression of immunosuppressive genes and on cytotoxic T lymphocyte activity against melanoma. It includes generation of specific knockout melanoma cells, and evaluation of ULK inhibition on T-cell mediated tumor killing in vitro. Aim 2 will define the role of ULK1 in ICB- mediated immune responses in melanoma. The effects on melanoma tumor growth of genetic or pharmacological inhibition of ULK1 in combination with immune checkpoint inhibitors will be examined in vivo. Additionally, the expression and activation of ULK1 will be correlated to clinical responses in melanoma patients receiving ICB therapy. The results of this project will advance our understanding on how specific IFN? signaling events affect the success of immune responses against melanoma and will provide the basis for novel targeted approaches involving combination of specific regulators of IFN? signaling with immune checkpoint inhibitors for the treatment of melanoma.